Approximately half the proteins present in cells are associated with lipid bilayers, and they arguably perform some of the most interesting and important functions in the cell. For example, ion channels control the cellular environment and facilitate nerve signaling, while membrane receptors are critical for the control of cellular metabolism. Macromolecules that are key elements in viral infection and the immune response ate also membrane proteins. Unfortunately, relatively little is known regarding the structures, molecular operation and assembly of membrane proteins. This is primarily a result of the fact that approaches that can yield structural information on water-soluble proteins such as crystallography and high resolution NMR generally fail when applied to membrane proteins. The is potentially an enormous payoff when such structures are understood, because of the potential to design new pharmaceuticals that target specific structures and processes in these proteins. This structural information will also facilitate a molecular understanding of numerous genetic diseases. Presently, there are no voltage-gated channels where the molecular events leading to gating have been clearly elucidated, and the objective of the proposed research is to define the molecular mechanisms that lead to voltage-gating and regulation of two membrane ion channels. The structure and gating mechanism of alamethicin will be studied, a small peptide that produces a voltage-dependent conductance in membranes. It is a model for larger voltage-gated channels, and it also belongs to a wider group of membrane active peptides that have important antibiotic activities. A second protein that will be studied is phospholemman. This protein is found in the myocardium and it is the major sarcolemmal substrate of cyclic AMP dependent protein kinase A and protein kinase C. Phospholemman is an 8 KDa transmembrane protein that forms a voltage-activated anion channel. By characterizing the structure and molecular gating of these channels, it is anticipated that fundamental information regarding the nature of membrane ion transport and the structural changes associated with gating and channel regulation will be obtained. The proposed work will make use of spectroscopic techniques such as EPR, high resolution NMR and solid-state NMR to define the structures of these channels and the changes associated with gating and covalent modification. Spin-probes will be incorporated into these channels for EPR experiments using synthetic techniques and site-directed mutagenesis.